The present invention is related to the technical field of preparing and using soluble high molecular-weight chitosan.
Low water solubility has restricted applications of chitin (FIG. 1A), which resulted in the expanded use of its water-soluble derivatives chitosan (FIG. 1B). Chitosan, which is produced by deacetylation of chitin, offers many biomedical advantages, and has wide applications in the pharmaceutical and biomedical areas. The solubility of chitosan in water is limited to pH values lower than 6, wherein the glucosamine moiety is converted to R—NH3+ soluble form by the protonation of amine group. Although it was soluble at pH below 6, chitosan dispersions suffer from aggregation. The formation of these aggregates is related to interactions between the hydrophobic parts in different chitosan molecules as well as hydrogen bonding between chitosan molecules. The aggregation of chitosan molecules decreases their interactions with water.
Several studies were conducted in attempt to enhance solubility of chitosan in water. The main methods have focused on preparation of small molecular-weight oligomers, changing the degree of deacetylation, and chemical modification such as the preparation of carboxymethyl chitosan.
U.S. Pat. No. 6,716,970 has disclosed a method of acetylation of chitosan to produce water-soluble derivative at pH values between 6 and 8. Patent CN 102786607 teaches a method of producing water-soluble chitosan oligosaccharides by hydrolysis using hydrogen peroxide at increase temperatures. Patent CN 02321194 describes facilitation of chitosan hydrolysis when subjected to ultrasonication. Patent WO2014014370A2 has disclosed a method for obtaining aqueous solution of chitosan. Another patent CN 103113490A has described preparation of water-soluble chitosan phosphate derivatives to be used as metal corrosion inhibitor. Korean patent 441270 has described the preparation of water-soluble free amine chitosan (1000-100.000 Da) by treatment of acidic solution of chitosan with trialkylamine with addition of organic solvent to remove the organic acid, followed by a purification step using activated carbon/ion exchange column. The prepared polymer is claimed to be non-toxic and biocompatible. US Patent Publication 20100040694 relates to preparation of low-molecular weight water-soluble chitosan nanoparticles for gene delivery with folic acid conjugates. Another patent WO2007013717 has disclosed preparation of high quality water-soluble chitosan oligosaccharide (1000-11.000 Da) using ultrafiltration by freeze drying, introduction of organic solvent, and vacuum drying.
However, the above described methods suffer from several disadvantages, which include reproducibility issues even for skilled people, difficulties in scaling up for industrial applications, significant time consumption, the requirement of large amounts of solvents, and finally changes in the important characteristics of the prepared polymers such as reduction of the molecular weight of the polymers. The disclosed methods also change other important physicochemical properties such as viscosity, degree of deacetylation, particle size, and density, which affect many practical applications in the pharmaceutical field and the food industry. Chemical derivatization in many studies has the drawback in complex multi-stage procedures and the use of organic solvents which are harmful for the health and environment. In addition, the chitosan derivatives do not maintain biocompatibility and stability compared to non-modified chitosan.
Supercritical fluid technique offers many advantages over other techniques. It decreases consumption of organic solvents and is thus a greener technology. The procedure can be carried at moderate temperatures, with the capability to control particle size and morphology. This technology has wide applications, including extraction, particle generation, preparation of inclusion complexes, and chemical reactions that require high speed. CO2 is the most commonly used substance in supercritical fluid technology. It offers many advantages over other substances. It can be used at moderate processing parameters, with a critical temperature of 31.1° C. and a critical pressure of 73.8 bar. It is inflammable, not toxic, and available at low cost. Patent PL 198876 teaches a method of obtaining dry chitosan membrane using supercritical fluid technology.
The presence of primary amino and two hydroxyl groups in chitosan allows the possibility of chemical modification in the structure. Although chitosan is insoluble in supercritical fluid CO2, solubility of CO2 is expected to be high in chitosan. This property leads to swelling of the polymeric chains, causing plasticization, which in turn can increase segmental and chain mobility, causing an increase in the inter chain distance. The main advantage in this state is that molecular weight of the polymer is of little influence on the swelling. Another advantage is in the enhancement of CO2 diffusion inside the polymeric chain, which facilitates interaction between CO2 and the polymer.
However, there have been limited studies concerning polymeric modification of chitosan using supercritical fluid CO2. There is still a need for a method to increase chitosan's water solubility at median pH values.